Fig. 7: TMEM165 mediates H⁺ inward and Ca²⁺ outward currents.

a Voltage step protocol employed for current recordings. b Examples of macroscopic current recordings conducted for TMEM165-WT-mCherry (green), TMEM165-R126C-mCherry mutant (R126C, red), and mCherry control (black) transiently expressed in HEK293 cells. Measurements were performed using indicated pH and Ca²⁺ concentrations in the pipette/bath. c Current densities plotted against respective voltages for TMEM165-WT-mCherry (left, green squares, n = 6) and TMEM165-R126C-mCherry (right, red triangles, n = 6) and the control (black circles, n = 12). d Representative (n = 3) live images of HEK293 cells transiently transfected with TMEM165-WT- or TMEM165-R126C-mCherry. Arrows, plasma membrane; scale bars, 10 µm. e Examples of macroscopic current recordings conducted for TMEM165-WT-mCherry using indicated pH and Ca²⁺ concentrations in the pipette/bath. f Quantitative analyses of current densities (I/C_m) plotted against respective voltages for TMEM165-WT-mCherry at indicated pH and Ca²⁺ concentrations in the pipette/bath solution with asymmetrical Ca²⁺ and pH (green squares, n = 6) or asymmetrical Ca²⁺ and symmetrical pH (blue reverted triangles, n = 4), symmetrical Ca²⁺ and symmetrical pH (magenta circles, n = 3), and symmetrical Ca²⁺ and asymmetrical pH (mustard circles, n = 4). g, h Current densities plotted against respective voltages for TMEM165-WT-mCherry at indicated pH and Ca²⁺ concentrations in the pipette/bath solution with asymmetrical Ca²⁺ and pH with (green squares, n = 6) and without (mustard circles, n = 9, right) Mg²⁺ or symmetrical Ca²⁺ and asymmetrical pH without Mg²⁺ (cyan circles n = 7, left). Bars, mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 as analyzed by two-tailed, homoscedastic t-test using GraphPad Prism 10.2.3.